1. Field of the Invention
The present invention relates to equine amniotic fluid-derived multipotent stem cells (eAF-MSCs) and a preparation method thereof. More specifically, the present invention relates to equine amniotic fluid-derived multipotent stem cells which exhibit all negative immunological characteristics with respect to the human markers CD19, CD20, CD28, CD31, CD34, CD38, CD41a, CD62L, CD62P and CD200, and exhibit all positive immunological characteristics with respect to the human markers CD44, CD90 and CD105, and have the ability to differentiate into ectoderm, mesoderm or endoderm-derived cells and the ability to maintain an undifferentiated state for 14 passages or more.
2. Description of the Related Art
Biotechnology in the 21st century presents the possibility of new solutions to food, environmental and health problems, with the ultimate object of promoting human prosperity. In recent years, the technology of using stem cells has been considered as a new way to treat incurable diseases. Previously, organ transplantation, gene therapy, etc., were considered for the treatment of incurable diseases. However, their efficient use has not been achieved due to factors such as rejection by the immune system, the short supply of organs, and an insufficient knowledge of disease genes
For these reasons, there has been an increased interest in the study of stem cells and it has been recognized that totipotent stem cells, having the ability to form all the organs by proliferation and differentiation, can not only treat most diseases but can also fundamentally heal organ injuries. Also, many scientists have suggested clinical applicability of stem cells for the regeneration of all the organs and the treatment of incurable diseases, including Parkinson's disease, various cancers, diabetes and spinal damages.
Stem cells refer to cells having not only self-replicating ability but also the ability to differentiate into at least two cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells (MSCs).
Totipotent stem cells are cells having totipotent properties capable of developing into one perfect individual, and these properties are possessed by cells up to the 8-cell stage after the fertilization of an oocyte and a sperm. When these cells are isolated and transplanted into the uterus, they can develop into one perfect individual. Pluripotent stem cells, which are cells capable of developing into various cells and tissues derived from the ectodermal, mesodermal and endodermal layers, are derived from an inner cell mass located inside of blastocysts generated 4-5 days after fertilization. These cells are called embryonic stem cells and can differentiate into various other tissue cells but not form new living organisms.
Multipotent stem cells were first isolated from adult bone marrow (Y. Jiang et al., Nature, 418:41, 2002), and then also found in other various adult tissues (C. M. Verfaillie, Trends Cell Biol., 12:502, 2002). In other words, although bone marrow is the most widely known source of stem cells, the multipotent stem cells were also found in the skin, blood vessels, muscles and brains (J. G. Toma et al., Nat. Cell Biol., 3:778, 2001; M. Sampaolesi et al., Science, 301:487, 2003; Y. Jiang et al., Exp. Hematol., 30:896, 2002). However, stem cells are very rarely present in adult tissues, such as bone marrow, and such cells are difficult to culture without inducing differentiation, and thus difficult to culture in the absence of specifically screened media.
Equine is a mammal belonging to the Equidae (Genus Equus), and very familiar with humans, along with dogs or cats. Equine industries, including the horse racing industry, are growing worldwide. In the horse racing industry, because the value depends on the horse's pedigree, injuries of racehorses with a good pedigree are a major problem. Thus, the development of cell therapy has attracted attention. Considering the size of racehorses, it was a main technical issue to reliably supply a large number of cells.
Therefore, isolation and characterization of stem cells derived from various equine tissues are important issues in the stem cell field. Conventionally, there have been many attempts to isolate stem cells from human and mouse tissues. However, cell therapy for the treatment of muscle, bone, cartilage, tendon or muscle of horses, in particular racehorses, has not yet been actively studied.
There is a report on the isolation of stem cells from equine adipose tissues (Armando de Mattos Carvalho et al., Veterinary Immunology and Immunopathology, 132:303, 2009), but, unlike other mammals, horses do not have much adipose tissue, and thus there is a difficulty in obtaining them. In addition, stem cell isolation from adipose tissue is a painful invasive procedure. Isolation of stem cells from equine bone marrow is also an invasive procedure, as in the procedure of the adipose tissue. Due to the nature of racehorses, there is also a limitation in the isolation of stem cells from adipose tissue or bone marrow.
At the present technical level, to use adult stem cells as the cell therapeutic agent, it is required to standardize the culture conditions under which the undifferentiated state can be maintained. In addition, since adult stem cells isolated from tissues are present in the mixed state of various kinds of cells, technology is sought that is capable of culturing homogeneous adult stem cells on a mass scale. In particular, the method for isolating adult stem cells from tissues or blood generally includes, for example, cell sorting utilizing antibodies for numerous surface antigens. However, this method has the limitation that the surface antigens for adult stem cells should be understood, and further, its use is greatly restricted by the problems that the common surface antigen (hereinafter, referred to as “marker”) for adult stem cells is not known. Also, markers for adult stem cells are not variously developed, and further, even the known markers show different degrees of expression depending on the differentiation state. Additionally, the sorting equipment is expensive.
Further, adult stem cells may possibly be mixed with other mononuclear cells and other stem cells in amniotic fluid. However, such mixed cell culture conditions may result in a difference in the distribution degree of nutrients, thereby leading to the heterogeneity of cell differentiation state. In the end, the problem that the cells cannot be produced as the homogeneous cell population serves as the fatal disadvantage that when they are used as the therapeutic agent, the actual effect may be different from the intended effect. Therefore, there is an urgent need for development of the effective culture technology which can provide homogeneous adult stem cells in a large quantity.
Accordingly, the present inventors first isolated stem cell populations with improved homogeneity from equine amniotic fluid as a new source of cell supply. They found that these cells exhibit more rapid and steady self-renewal ability (growth ability) than other stem cells, immunological characteristics of multipotent stem cells, and excellent multilineage differentiation capability, in particular, outstanding osteogenic differentiation capability, thereby completing the present invention.